| 摘要: |
| 【目的】研究大口黑鲈双RNA病毒（largemouth bass birnavirus，LBBV）VP2蛋白的抗原性，为后续LBBV疫苗研究提供基础材料。【方法】PCR扩增LBBV VP2基因，构建重组质粒pET-VP2，PCR和测序鉴定后转入大肠杆菌中进行诱导表达和纯化。以纯化的重组VP2蛋白为抗原，皮下多点注射免疫新西兰大白兔，耳中央动脉采血并分离血清，通过间接ELISA方法测定多克隆抗体效价；利用Ni NTA镍离子亲和层析纯化抗体，用BCA法测定纯化后抗体的浓度。通过Western blot和间接免疫荧光鉴定VP2多克隆抗体特异性；采用固定病毒-稀释血清法测定血清的中和效价。【结果】PCR扩增获得了1 266 bp的VP2基因片段。PCR和测序鉴定结果表明，重组质粒pET-VP2构建成功。VP2重组蛋白的诱导表达和纯化试验结果获得了64 ku的重组蛋白，符合预期大小。成功制备了VP2蛋白的多克隆抗体，其质量浓度为10 mg/mL，效价为1∶1 024 000，可特异性识别LBBV VP2蛋白；血清中和试验结果显示，VP2多抗中和抗体效价为1∶64。【结论】成功获得了大肠杆菌表达的LBBV重组VP2蛋白，制备了抗VP2蛋白的多克隆抗体，该多抗对LBBV具有中和作用。 |
| 关键词: 大口黑鲈 大口黑鲈双RNA病毒 VP2蛋白 多克隆抗体 |
| DOI：10.13207/j.cnki.jnwafu.2025.07.003 |
| 分类号: |
| 基金项目:广州市重点研发计划项目（2024B03J1263）；中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金项目（2024XT0502） |
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| Prokaryotic expression of largemouth bass birnavirus VP2 protein and preparation of polyclonal antibody |
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ZHANG Qiushuang, MA Baofu, ZHENG Guo, LIN Qiang, LIANG Hongru, NIU Yinjie, LUO Xia, LI Ningqiu, FU Xiaozhe
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Prokaryotic expression of largemouth bass birnavirus VP2 protein and preparation of polyclonal antibody
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| Abstract: |
| 【Objective】This research aimed to study the antigenicity of the VP2 protein of largemouth bass birnavirus (LBBV),to provide basic materials for the subsequent research on LBBV vaccine.【Method】The LBBV VP2 gene was amplified by PCR,and the recombinant plasmid pET-VP2 was constructed,which was identified by PCR and sequencing,and then transferred into Escherichia coli (E. coli) for induced expression and purification.The purified recombinant VP2 protein was used as the antigen to immunize the New Zealand White rabbits with subcutaneous multipoint injection.Blood was collected from the central artery of the rabbit ear and serum was separated,and the polyclonal antibody potency was determined by indirect ELISA.The antibody was purified by Ni-NTA nickel ion affinity chromatography,and the concentration of the purified antibody was determined by BCA method.The specificity of VP2 polyclonal antibody was identified by Western blot and indirect immunofluorescence,and the neutralization potency of serum was determined by fixed virus-diluted serum method.【Result】With PCR amplification,a VP2 gene fragment of 1 266 bp was obtained.PCR and sequencing identification results showed that the recombinant plasmid pET-VP2 was successfully constructed. 64 ku of recombinant protein was obtained from the induced expression and purification of VP2 recombinant protein, which was in line with the expected size.The polyclonal antibody to VP2 protein was successfully prepared with a mass concentration of 10 mg/mL and a potency of 1∶1 024 000,which can specifically recognize LBBV VP2 protein.The results of serum neutralization experiments showed that the potency of the neutralizing antibody to VP2 polyantibody was 1∶64.【Conclusion】In this research,with LBBV as the research object,the recombinant VP2 protein expressed in E. coli was successfully obtained,and the polyclonal antibody against the LBBV VP2 protein was then successfully prepared.The polyclonal antibody had a neutralising effect on LBBV. |
| Key words: largemouth bass largemouth bass birnavirus (LBBV) VP2 protein polyclonal antibody |
